Article
pubs.acs.org/Langmuir
Mixtures of Lecithin and Bile Salt Can Form Highly Viscous Wormlike
Micellar Solutions in Water
Chih-Yang Cheng,† Hyuntaek Oh,§ Ting-Yu Wang,‡ Srinivasa R. Raghavan,§ and Shih-Huang Tung*,†
†
Institute of Polymer Science and Engineering and ‡Instrumentation Center, National Taiwan University, Taipei 10617, Taiwan
§
Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland 20742, United States
S Supporting Information
*
ABSTRACT: The self-assembly of biological surfactants in
water is an important topic for study because of its relevance to
physiological processes. Two common types of biosurfactants
are lecithin (phosphatidylcholine) and bile salts, which are
both present in bile and involved in digestion. Previous studies
on lecithin−bile salt mixtures have reported the formation of
short, rodlike micelles. Here, we show that lecithin−bile salt
micelles can be further induced to grow into long, flexible wormlike structures. The formation of long worms and their resultant
entanglement into transient networks is reflected in the rheology: the fluids become viscoelastic and exhibit Maxwellian behavior,
and their zero-shear viscosity can be up to a 1000-fold higher than that of water. The presence of worms is further confirmed by
data from small-angle neutron and X-ray scattering and from cryo-transmission electron microscopy (cryo-TEM). We find that
micellar growth peaks at a specific molar ratio (near equimolar) of bile salt:lecithin, which suggests a strong binding interaction
between the two species. In addition, micellar growth also requires a sufficient concentration of background electrolyte such as
NaCl or sodium citrate that serves to screen the electrostatic repulsion of the amphiphiles and to “salt out” the amphiphiles. We
postulate a mechanism based on changes in the molecular geometry caused by bile salts and electrolytes to explain the micellar
growth.
1. INTRODUCTION
The self-assembly of biological surfactants in water has been
intensely investigated by scientists over the past several
decades. Two common classes of such surfactants are
phospholipids and bile salts. A typical phospholipid is lecithin,
i.e., phosphatidylcholine, which is a zwitterionic molecule with a
headgroup having a positively charged choline and a negatively
charged phosphate (Figure 1a). In water, lecithin tends to selforganize into bilayer membranes and in turn into vesicles. Bile
salts are amphiphiles synthesized in the body within the liver.
They have a characteristic steroid structure with a rigid,
nonplanar, fused ring and a carboxylic acid group that may be
conjugated with amino acids such as taurine and glycine.
Different bile salts contain different numbers (one to three)
and positions of hydroxyl groups attached to the rings (Figure
1b). Unlike conventional amphiphiles that have a hydrophilic
head and hydrophobic tail(s), bile salts are facial amphiphiles
with polar and nonpolar faces.1 Because of this facial structure,
bile salts in water form unusual micelles in which the molecules
are packed back-to-back with low aggregation numbers (5−
10).2
The aqueous self-assembly of lecithin/bile salt mixtures is of
general interest due to its physiological relevance as well as its
biochemical and biomedical applications.1,3−8 While lecithin
alone forms vesicles, these vesicles are unstable because the
bulky hydrophobic tails of the lipid inhibit its solubility in
water. Bile salts are much more soluble in water, and in small
amounts, they can intercalate into lecithin vesicles and stabilize
© 2014 American Chemical Society
Figure 1. Molecular structures of (a) soybean lecithin and (b) bile
salts used in this study.
Received: June 18, 2014
Revised: August 14, 2014
Published: August 14, 2014
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forces between bile salt anions as well as the Hofmeister effect
that “salt out” the bile salts.28 We have postulated a mechanism
to explain why the above conditions are required for the growth
of worms.
Our finding that lecithin/bile salt mixtures can form worms
in water is interesting and potentially important. These findings
could be relevant for understanding the physiological roles of
the different components in bile. It could have implications for
diseases of the liver, pancreas, or gall bladder, such as
hypercholesterolemia and gallstone formation.29,30 Moreover,
phospholipid-based gels or viscoelastic fluids have recently been
evaluated as candidates for delivering drugs across skin.31,32 For
instance, lecithin-based reverse worms in oil have been shown
to enhance the dermal permeation of drugs.31 In a similar vein,
lecithin/bile salt based worms in water may also be useful as
transdermal carriers of active ingredients that could be released
in a controlled manner. In other words, the biocompatibity and
viscoelasticity of the system can make it useful in medical or
cosmetic formulations.
these structures. Accordingly, lecithin−bile salt vesicles have
been examined in the context of lipid−protein interactions9 and
the enzymatic hydrolysis of phospholipids.10 At higher
amounts, bile salts and lecithin form mixed micelles.
Physiologically, mixed micelles of lipids and bile salts are
known to play a crucial role in the digestion, intestinal
absorption, and transport of dietary fats, including cholesterol.1,3,11 From an application standpoint, mixed micelles and
vesicles containing bile salts and lecithin have been employed as
carriers for delivering drugs across the intestinal walls.12,13
The self-assembly of lecithin/bile salt mixtures in water is
known to be dependent on the molar ratio of the two
species,14,15 the total lipid concentration,16 and the ionic
strength.17 These mixtures form micelles at a molar ratio of bile
salt to lecithin ∼1. Initial studies suggested that the micelles are
disklike in shape.18 However, later evidence from light
scattering,19 small-angle neutron scattering (SANS),15,20 cryotransmission electron microscopy (cryo-TEM),14 and other21
techniques have shown that the micelles are cylindrical in
shape. A couple of studies have suggested that the cylinders can
sometimes grow into long chains.14,15 Cylindrical micellar
chains that are long and flexible are termed wormlike micelles
(“worms” for short). Much like polymer chains, worms are
expected to entangle into a transient network, transforming the
solution from a Newtonian, low-viscosity state into a highly
viscous and viscoelastic state.22−24 The zero-shear viscosity η0
of semidilute worms (<10 wt %) can be several orders of
magnitude higher than that of water.25 However, to our
knowledge, highly viscous worms of lecithin/bile salt in water
have not been reported so far. This suggests that the cylindrical
lecithin/bile salt micelles prepared in previous studies are
relatively short and thereby unable to entangle with one
another.
Apart from aqueous systems, mixtures of lecithin and bile
salts have also been studied in nonpolar organic solvents (oils),
where they show interesting self-assembly. Lecithin alone in oils
forms reverse spherical or ellipsoidal micelles.26 Bile salts are
insoluble in oils, but they can be dissolved in the presence of
lecithin. When bile salts are added to lecithin reverse micelles,
the micelles grow into long flexible cylinders, which are termed
reverse wormlike micelles. The mechanism for this transition is
believed to involve binding of the bile salts to the phosphate
groups on lecithin, which then alters the effective geometry of
the molecules so as to favor long cylinders.26,27 Remarkably, the
zero-shear viscosity η0 of these reverse worms can be 5 orders
of magnitude higher than that of the parent oil. Thus, it is
known that lecithin/bile salt can form long reverse worms in
oil; however, “normal” worms of the same in water have not
been reported yet.
In this work, we have succeeded in finding a set of conditions
that result in highly viscous semidilute solutions of lecithin/bile
salt in water. We confirm via rheology, scattering techniques,
and cryo-TEM that the high viscosities are due to the formation
of wormlike micelles. We show that worms can be obtained
using four different bile salts (Figure 1b): the trihydroxy bile
salts, sodium cholate (SC) and sodium taurocholate (STC),
and the dihydroxy bile salts, sodium deoxycholate (SDC) and
sodium taurodeoxycholate (STDC). The formation of worms is
maximized at a specific molar ratio of bile salt:lecithin, and in
turn the viscosity reaches a peak at this molar ratio.
Additionally, the formation of worms also requires a background electrolyte at sufficient levels. This background
electrolyte provides an ionic strength to screen the repulsive
2. EXPERIMENTAL SECTION
2.1. Materials. Soybean lecithin (95% purity) was purchased from
Avanti Polar Lipids, Inc. The bile salts sodium cholate (SC, >99%),
sodium deoxycholate (SDC, >97% purity), sodium taurocholate (STC,
>97%), and sodium taurodeoxycholate (STDC, >95%) were
purchased from Sigma-Aldrich. Sodium chloride (NaCl, >99.7%),
calcium chloride dihydrate (CaCl2), and sodium citrate dihydrate
(>99.9%) were purchased from J.T. Baker. Sodium sulfate dihydrate
(Na2SO4, >99.3%) was purchased from Macron Chemicals. Iron
chloride (FeCl3, >98%) was purchased from Alfa Aesar. All chemicals
were used as received.
2.2. Sample Preparation. Lecithin and bile salts were dissolved in
methanol to form 200 mM stock solutions. The molar ratio of bile salt
to lecithin was adjusted by mixing appropriate amounts of the stock
solutions. After mixing, methanol was removed by drying the samples
in a vacuum oven at 50 °C for 48 h. The final samples with desired
amphiphile and electrolyte concentrations were obtained by adding
proper amounts of deionized water and electrolytes, followed by
stirring and sonication until the solutions were well mixed.
2.3. Rheology. We performed steady and dynamic rheological
experiments on an AR2000EX stress controlled rheometer (TA
Instruments). Cone-and-plate geometry (40 mm diameter with 4°
cone angle) was used for all samples. The plate was equipped with
Peltier-based temperature control, and all samples were studied at 25
± 0.1 °C. We utilized a solvent trap to minimize the evaporation of
water. Dynamic frequency spectra were conducted in the linear
viscoelastic regime of the samples, as determined from dynamic strain
sweep measurements. For steady-shear experiments, sufficient time
was allowed before data collection at each shear rate to ensure that the
viscosity reached its steady-state value.
2.4. Cryogenic Transmission Electron Microscopy (CryoTEM). Specimens for cryo-TEM were prepared with a FEI Vitrobot
Mark IV vitrification system at 22 °C and 100% relatively humidity.
Samples were applied onto grids with porous carbon films. After threetime blotting processes, the grids were immediately plunged into liquid
ethane and then stored in liquid nitrogen before imaging. TEM
imaging were conducted on a FEI Tecnai G2 transmission electron
microscope operated at a voltage of 120 kV.
2.5. Small-Angle Neutron and X-ray Scattering (SANS and
SAXS). SANS measurements were made on the NG-7 (30 m)
beamline at NIST in Gaitherburg, MD. Neutrons with a wavelength of
6 Å were selected. Two sample−detector distances were used to probe
a wide range of wave vectors from 0.0035 to 0.5 Å−1. Deuterated water
(D2O) instead of protonated water was used as solvent to enhance the
scattering contrast. SAXS measurements were conducted on the
BL23A1 beamline in the National Synchrotron Radiation Research
Center (NSRRC), Taiwan.33 A monochromatic beam of λ = 0.83 Å
was used. The scattering patterns were collected on a Pilatus 1M-F
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detector over a q range from 0.01 to 0.4 Å−1. All the samples were
studied at 25 °C. The spectra are shown as plots of the absolute
intensity I versus the wave vector q = 4π sin(θ/2)/λ, where θ is the
scattering angle and λ is the wavelength.
2.6. SANS and SAXS Modeling. Modeling of SANS and SAXS
data was conducted using the analysis package provided by NIST with
IGOR Pro software.34 For dilute solution of noninteracting scatterers,
the scattering intensity I(q) can be modeled in terms of the form factor
P(q) of scatterers
I(q) = cP(q) + B
respectively. α is the angle between the cylinder axis and the scattering
vector q. J1(x) is the first-order Bessel function and J0 is given by
J0 (x) = sin(x)/x
3. RESULTS AND DISCUSSION
3.1. Phase Behavior and Rheology. We first discuss the
effect of bile salt:lecithin molar ratio, denoted as B0, on the
rheological properties of lecithin/bile salt mixtures in water at
sufficient electrolyte concentration. The zero-shear viscosity η0
as a function of B0 for representative lecithin/SC mixtures at a
high NaCl concentration of 5 M is shown in Figure 2a. The
(1)
where c is the total concentration of scatterers and B is the
background.
For SANS data, we consider form factor model for flexible cylinder
with polydispersity radius. The form factor for semiflexible chains of
contour length L, Kuhn length b, and cross-sectional radius R can be
represented as the product of the excess scattering length density Δρ,
cross-section form factor PCS, and wormlike chain form factor
PWC:35,36
P(q) = Δρ2 PCS(q , R )PWC(q , L , b)
(10)
(2)
The cross-section form factor PCS can be approximated by the
following expression, which is valid for cylindrical chains
⎡ 2J (qR ) ⎤2
⎥
PCS(q , R ) = ⎢ 1
⎣ qR ⎦
(3)
where J1(x) is the first-order Bessel function given by
J1(x) =
sin x − x cos x
x2
(4)
For cylinders with polydispersity radius, the form factor should be
averaged over the radius distribution in the following manner
PCS(q , R 0) =
∫ f (R)PCS(q , R) dR
Figure 2. Zero-shear viscosity η0 as a function of the molar ratio of bile
salt to lecithin B0 for lecithin/SC mixtures in water. Lecithin
concentration is fixed at 100 mM, and NaCl concentration is 5 M.
(5)
where R0 is the average cylinder radius, the polydispersity in cylinder
radius f(R) is accounted for by a Schulz distribution
⎡ z + 1 ⎤z + 1
⎡
Lz
R⎤
exp⎢ − (z + 1) ⎥
f (R ) = ⎢
⎥
R0 ⎦
⎣ R 0 ⎦ Γ(z + 1)
⎣
lecithin concentration was fixed at 100 mM. At a B0 below 0.2,
the viscosity is almost as low as that of water. The low ratio of
bile salt is unable to alter the bilayer structure of lecithin so that
large aggregates are present in the solution and the solution is
turbid and of low viscosity (see photograph of typical sample
on the top left).17 As B0 increases above 0.2, the viscosity
increases rapidly and reaches a maximum at a molar ratio
denoted as B0max. The viscosity is about a 1000 times higher
than that of water. The sample is transparent and flows slowly
in the inverted vial as shown in the photograph, indicating a
transformation of bilayers into long cylindrical micelles. Such a
high viscosity can only be obtained in a narrow B0 range. After
B0max, the viscosity drops sharply. At B0 ∼ 1.3, the sample
becomes a thin, clear liquid with a viscosity close to that of
water, implying that the long micelles have been turned into
short ones. The four bile salts shown in Figure 1 exhibit very
similar trends in the η0−B0 relationship, and the B0max values for
SC, SDC, STC, and STDC are 0.9, 1.2, 0.8, and 1.0,
respectively. The zero-shear viscosities as a function of B0 for
SDC, STC, and STDC samples are shown in Figure S1 of the
Supporting Information.
Figure 3 shows the zero-shear viscosity η0 of the four bile
salts as a function of NaCl concentration [NaCl] in water at 25
°C. The lecithin concentration was fixed at 100 mM, and the
molar ratios of bile salt:lecithin were fixed at the B0max for each
bile salt. At B0max without the addition of NaCl, the solutions
are slightly cloudy and slightly viscous, indicating that bilayers
are still present. As [NaCl] increases, the solutions become
transparent and the viscosity is greatly enhanced, indicating a
(6)
where Γ is the gamma function and z is a parameter related to the
width of the distribution. The polydispersity index pd is given by
pd =
1
z+1
(7)
The form factor PWC for a wormlike chain with excluded-volume
interactions is detailed in the paper by Pedersen and Schurtenberger,36
where it was originally derived, and then modified by Chen et al.37
Because of the different origin of scattering contrast, the form factor
model of core−shell cylinder instead of flexible cylinder was used to fit
the SAXS data. The form factor of core−shell cylinders can be
represented as38
P(q) =
1
Vt
∫0
π /2
f 2 (q , α)sin α dα
(8)
where
⎛ qL
⎞ J (qR c sin α)
f (q , α) = 2(ρc − ρs )VcJ0 ⎜ c cos α⎟ 1
⎝ 2
⎠ qR c sin α
⎡ ⎛ Lc
⎤ J [q(R c + t ) sin α]
⎞
⎜
+ 2(ρs − ρsol )VJ
+ t ⎟ cos α ⎥ 1
t 0 ⎢q
⎠
⎣ ⎝2
⎦ [q(R c + t ) sin α]
(9)
where Rc is the core radius, Lc is the core length, t is the shell thickness.
Vc and Vt are the core volume and total volume, respectively. ρc, ρs,
and ρsol are scattering length density of core, shell, and solvent,
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Figure 3. Zero-shear viscosity η0 of lecithin−bile salt mixtures in water
as a function of NaCl concentration. The lecithin concentration is
fixed at 100 mM. B0s of SC, SDC, STC, and STDC are 0.9, 1.2, 0.8,
and 1.0, respectively. For STDC and SDC, the ends of the curves are
the phase boundaries above which the samples phase separate into two
liquid phases. For SC and STC, the samples remain homogeneous
until the saturated NaCl concentration ∼6 M is reached.
growth of the micelles. For STC, STDC, and SDC samples, the
viscosity reaches a plateau, whereas the viscosity of SC samples
increases monotonically. Only in the narrow B0 range shown in
Figure 2 and Figure S1 can we tune the electrolyte
concentration to observe this thickening phenomenon. In
other words, a specific molar ratio and a sufficient electrolyte
concentration are required for the formation of stable, long
flexible cylindrical micelles.
With further increase of NaCl concentration, dihydroxy bile
salt STDC and SDC samples phase separate into two liquid
phases at [NaCl] ∼ 1.5 and 2.5 M, respectively, while
trihydroxy STC and SC samples remain stable until [NaCl]
is saturated at ∼6 M. The number of hydroxyl groups on
steroid rings of bile salts must play a role in causing the
difference. It is known that the aqueous solubility of trihydroxy
bile salts is higher than that of dihydroxy ones and that the bile
salts conjugated with taurine groups show a lower solubility
than unconjugated ones.39 The solubility of the four bile salts is
therefore in the order of SC > STC > SDC > STDC. The
addition of NaCl screens the repulsive forces between the
negatively charged bile salt molecules and thus lowers their
solubility in water.40 A high [NaCl] may further decrease the
solubility of bile salts due to the interactions of Na+ and Cl−
ions with water molecules, known as the Hofmeister effect or
the “salting-out” effect.28 Since the solubilities of SDC and
STDC are lower than those of SC and STC, it is reasonable
that the former phase-separate at a lower [NaCl] than the
latter. Also, the [NaCl] for destabilizing the taurine-based
STDC sample is the lowest among the four bile salts.
The viscoelasticity of the lecithin−bile salt mixtures were
investigated by dynamic rheology. The elastic modulus (G′)
and the viscous modulus (G″) as functions of frequency ω for
trihydroxy SC and STC samples at their maximum viscosities
are shown in Figure 4. In each case, the G′ and G″ curves
intersect, and we see an elastic response at high frequencies and
a viscous one at low frequencies. This type of response is
characteristic of viscoelastic samples. We fit the data using a
Maxwell model with a single relaxation time, which is shown as
solid lines through the data:
G′(ω) =
Figure 4. Dynamic rheology data of (a) lecithin/SC mixture at B0 =
0.9 and [NaCl] = 6 M and (b) lecithin/STC mixture at B0 = 0.8 and
[NaCl] = 2 M in water at 25 °C. The lecithin concentrations of both
samples are 100 mM. The plots show the elastic modulus G′ and
viscous modulus G″ as functions of frequency ω. Fits to singlerelaxation time Maxwell model are shown as black solid lines.
G″(ω) =
1 + ω 2t R 2
(12)
Here Gp is the plateau modulus and tR is the relaxation time.
The data can be fitted reasonably well, especially for the SC
sample. In the cases of dihydroxy SDC and STDC (Figure S2
in the Supporting Information), G″ is larger than G′ over the
whole frequency range. This implies that the micellar chains are
less restricted by entanglements, and the samples therefore
remain liquid-like.
In addition to NaCl, we have also investigated the effects of
different types of electrolytes. The zero-shear viscosity data of
the four lecithin/bile salt mixtures at their B0maxs in the
presence of sodium sulfate (Na2SO4) and sodium citrate
(Na3C6H5O7) are shown in Figure 5a,b. Similar to NaCl, both
these electrolytes can effectively increase the viscosity. The
efficiency of the three sodium salts in increasing viscosity is in
the order Na3C6H5O7 > Na2SO4 > NaCl. This order is
consistent with the greater ionic strengths of sodium citrate and
sodium sulfate that more efficiently screen the repulsion
between bile salt anions at the same electrolyte concentrations.41 Another effect is that citrate and sulfate are known
for their ability to “salt out” solutes from water; i.e., they occur
on one end of the Hofmeister series.42 That is, citrate and
sulfate are strongly hydrated anions that reinforce the
interactions between water molecules and in turn mitigate the
interactions between water and various solutes. Note that NaCl
also shows a salting-out ability at much higher concentrations.
Thus, the effect of background electrolyte in our system is
consistent with the ability of specific ions to salt out solutes
from water. Also, we should point out that the ends of the
curves in Figure 5 are the phase boundaries beyond which the
samples become turbid. The electrolyte concentration at this
Gpω 2t R 2
1 + ω 2t R 2
Gpωt R
(11)
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3.2. SANS, SAXS, and Cryo-TEM Data. We utilized
SANS, SAXS, and cryo-TEM to elucidate the self-assembled
structures of lecithin−bile salt mixtures in aqueous solutions.
The lecithin concentration of the samples was fixed at 20 mM,
which is a relatively low value, so as to reduce the micellar
volume fraction and thereby reduce intermicellar interactions.
Representative SAXS data for an STDC sample at B0 = 1.0
without the addition of electrolytes are shown in Figure S3a of
the Supporting Information. The sample is slightly cloudy, and
the q−2 decay of intensity in the intermediate q range indicates
the presence of bilayer structures,35 which is confirmed by the
cryo-TEM image shown in Figure S3b where stacks of bilayers
can be clearly seen.
To probe the structures in the viscous regime, we obtained
SANS spectra on four samples corresponding to SC, SDC,
STC, and STDC, with their respective B0 = 0.9, 1.2, 0.8, and 1.0
and [NaCl] = 5.5, 0.5, 1.0, and 0.5 M. Note that SANS
measurements were conducted at a low concentration of
amphiphiles where the relative amount of bile salts available for
solubilizing the lecithin may decrease because the solutions
tend to maintain the concentration of free bile salts in water.47
The choice of lower [NaCl]s for SANS measurements is to
ensure the dilute solutions are stable. D2O was used as the
solvent for these samples. Neutron scattering originates from
the contrast in neutron density between the micelles
(protonated molecules) and the solvent (deuterated water),
and some water molecules may penetrate into the hydrophilic
parts of the micelles. The SANS data are shown in Figure 6a as
plots of scattered intensity I vs wave vector q. All four samples
exhibit a scaling of I ∼ q−1 at intermediate q, which is indicative
of cylindrical micelles.35,48 We fitted the data using the flexible
cylinder model with polydisperse radius (eqs 2−7), and the fits
are shown as solid lines through the data. The data are fitted
well by the model, indicating the presence of wormlike micelles
in the solutions. The fitting parameters from the modeling are
listed in Table 1. The radii of the mixed wormlike micelles are
nearly the same, ∼20 Å, independent of the type of bile salts.
Because of the limit of q range in this study and the low
amphiphile concentrations used in SANS measurements, the
contour lengths listed in Table 1 may not be the exact sizes of
the micelles and may not coincide with the trend of viscosity
measured at high amphiphile concentrations. The representative cryo-TEM micrograph of a STDC sample prepared in the
same manner is shown in Figure 7. The image reveals the
presence of long and flexible cylindrical micelles, in agreement
with the results from SANS.
Figure 6b shows the SAXS data on the same samples, and
these are very different from the SANS data. We can see clear
humps at q around 0.15 Å−1 that are absent in SANS, especially
for SDC, STC, and STDC. The contrast in X-ray scattering
results from the interactions of incident X-rays with the
electrons in the various species. In the present case, the micellar
solutions contain three domains with distinguishable electron
densities: the hydrophobic core of the micelles, the hydrophilic
shell of the micelles, and the solvent (H2O). Therefore, the
SAXS data are expected to reflect a core−shell structure for the
micelles, unlike the uniform structure extracted from SANS
data. We have fitted the SAXS data with the model for core−
shell cylinders (eqs 8−10). Fits to the model are shown as solid
lines, and these fit the data reasonably well. The fitting
parameters are listed in Table 1. For SDC, STC, and STDC
samples, the core radius is 8−10 Å and the shell thickness is
20−21 Å. The core is composed of flexible lecithin tails while
Figure 5. Zero-shear viscosity η0 of the four lecithin/bile salt mixtures
in the presence of varying electrolytes: (a) sodium sulfate (Na2SO4),
(b) sodium citrate (Na3C6H5O7), and (c) calcium chloride (CaCl2).
B0s are 0.9, 1.2, 0.8, and 1.0 for SC, SDC, STC, and STDC samples,
respectively.
boundary follows the order NaCl > Na2SO4 > Na3C6H5O7,
which is again consistent with the ionic strength and hydrated
anions reducing solubility, leading to phase separation at a
lower electrolyte concentration.
We also studied electrolytes with multivalent cations, i.e.,
calcium chloride (CaCl2) and iron chloride (FeCl3). From the
viscosity data shown in Figure 5c, the efficiency of divalent Ca2+
in increasing viscosity is much higher than that of monovalent
Na+; i.e., the viscosity maximum is reached at a much lower
CaCl2 concentration, ∼0.1 M, for all four bile salts. The phase
separation boundaries also appear at low CaCl2 concentrations,
in agreement with previous reports that calcium bile salts are in
general less soluble in water than their sodium counterparts.43
When iron chloride (FeCl3) is added to the mixtures, a trace of
trivalent Fe3+ immediately turn the solutions into turbid, and
no significant increase in viscosity was observed (data not
shown). These results reflect a combination of two aspects.
First, cations of higher valency are more effective at screening
electrostatic repulsions of bile salt anions in solution.44 Another
possibility is that some multivalent cations, such as Ca2+, may
interact strongly with the negatively charged phosphate groups
of lecithin and cause aggregation of lecithin molecules.45,46
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Figure 7. Cryo-TEM image of lecithin/STDC sample at B0 = 1.0 and
[NaCl] = 0.5 M. The sample is found to contain long wormlike
micelles.
We also utilized the indirect Fourier transformation (IFT)
technique to analyze the SAXS and SANS data. IFT gives the
pair-distance distribution function p(r) corresponding to each
I(q) data, and these are shown in Figure 8. The p(r)
corresponding to the SANS data for all four mixed micelles
show an asymmetrical shape (Figure 8a), with an inflection
point followed by a decrease to zero. This p(r) function is
characteristic of cylindrical micelles.49 The inflection point gives
the approximate cylinder diameter, while the point where p(r)
meets the r-axis is an estimate for the average length of the
cylinders. Note that if the cylinders are rigid, p(r) should show
a linear decrease. The shoulders at r ∼ 100 Å are possibly due
to the flexible nature of the micelles. Figure 8b shows the p(r)
functions corresponding to the SAXS data. At low r, the p(r) of
SDC, STDC, and STC samples exhibit pronounced maxima
and minima, which are regarded as features of core−shell
structures.50−52 Similar to p(r) from SANS, asymmetric
decreases to zero can be seen for all the four bile salts. The
inflection point between the first maximum and minimum
reflects the cross-sectional sizes of the hydrophobic cores while
the inflection point after the second maximum gives the
cylinder diameter. The lengths of the cylinders can similarly be
estimated from the largest r values where p(r) approaches zero.
Note that for the SC sample, due to its indistinctive core−shell
feature, the second maximum almost disappears, and the p(r)
profile is similar to that of a regular cylinder.
It has been shown that at a fixed NaCl concentration the
viscosity reaches a maximum and then decreases with
Figure 6. (a) SANS and (b) SAXS data for lecithin/bile salt mixtures
at 25 °C. The lecithin concentration is fixed at 20 mM for all the
samples. B0 values are 0.9, 1.2, 0.8, and 1.0, and NaCl concentrations
are 5.5, 0.5, 1.0, and 0.5 M for SC, SDC, STC, and STDC samples,
respectively. Model fits are shown as solid lines through each data.
the shell is composed of lecithin headgroups, bile salts, and
maybe some water with electrolyte ions. Both the core and shell
sizes are larger for the SC sample, and it should be also noted
that for the SC sample the hump at q ∼ 0.15 Å−1 is nearly
absent, indicating a loss of core−shell characteristics. The larger
cross-sectional size of SC mixed micelles and the absence of the
hump from the SAXS data are likely due to the high NaCl
concentration (5.5 M) in this sample. Such a high NaCl
concentration may alter the electron density in the hydrophilic
shell of the micelles as well as in the solvent.
Table 1. Fitting Parameters from SANS and SAXS Modeling
SANS
SAXS
av radius
Kuhn length
contour length
polydispersity
core radius
shell thickness
core length
sample
R0 (Å)
b (Å)
L (Å)
pd
Rc (Å)
t (Å)
Lc (Å)
SC
SDC
STC
STDC
19.6
20.4
21.1
19.4
±
±
±
±
0.14
0.17
0.17
0.16
12.7
8.1
9.4
9.9
22.4
21.4
21.5
19.9
196.4
226.3
311.6
680.6
±
±
±
±
2.4
1.5
13.1
57.5
1678.1
2865.7
2607.5
579.3
18.1
212.5
251.5
4.3
10226
4647.1
368.5
1599.9
7752.2
±
±
±
±
2.8
1.1
5.1
5.4
dx.doi.org/10.1021/la502380q | Langmuir 2014, 30, 10221−10230
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Figure 9. SANS data of lecithin/STC samples at B0 ≥ B0max (∼0.8).
The lecithin concentration is 20 mM, and the NaCl concentration is
1.0 M. The intensity at low q greatly decreases with increasing B0,
indicating long cylindrical micelles are transformed into short ones.
presence of long wormlike micelles significantly enhances the
viscosity and imparts viscoelastic character to the samples. In
the current section, we will discuss why these conditions are
crucial for the micellar growth.
As described in the Introduction, in aqueous solution, the
addition of bile salt transforms lecithin vesicles or bilayers into
cylindrical mixed micelles. The shape of self-assembled
structures is governed by the effective geometry of the
amphiphiles, which can be simply expressed by the critical
packing parameter p = vtail/(ahgltail), where vtail is the volume of
tail, ahg is the head group area, and ltail is the tail length.54 The
transition from bilayers to cylindrical micelles implies a
reduction in packing parameter from ca. 1 to 1/2. In the
lecithin/bile salt case, this is achieved by an increase in effective
headgroup area due to the insertion of bile salts adjacent to the
lecithin headgroups within the self-assembled aggregates, as
illustrated in Figure 10. It is most likely that the carboxylate
(SC and SDC) or sulfonate (STC and STDC) groups of bile
salts expose to water and provide the major contribution to
stabilizing the aggregates. We expect bile salts to utilize the
hydroxyl groups on their steroid rings to form hydrogen bonds
with the phosphates on lecithin. Since bile salts are facial
amphiphiles, a back-to-back arrangement of bile salts is
favorable because this form can prevent the hydrophobic
faces of bile salts from contacting water. Such a model is
consistent with that proposed by Madenci et al.,15 and it is also
consistent with the core−shell structure deduced from SAXS
analysis.
Although lecithin/bile salt cylindrical micelles can be formed,
the length of such micelles (without sufficient background
electrolytes) is limited and unable to thicken the solutions. Why
is the length limited? This can probably be attributed to the
distribution of lecithin and bile salts in the micelles. It has been
suggested that the distribution of lecithin and bile salt
molecules in mixed micelles is not uniform.15 The two-tail
lecithin molecules with a nearly cylindrical molecular shape
prefer to locate in the low-curvature cylindrical bodies while
bile salts that generally form highly curved small micelles in
water are expected to form the hemispherical end-caps of the
cylinders, as shown by the schematic in Figure 10. In other
Figure 8. Pair-distance distribution functions p(r) obtained by IFT
analysis of the SANS and SAXS data shown in Figure 6.
increasing B0, and the samples remain homogeneous and
transparent, as shown in Figure 2. The SANS data of STC
samples with B0 = 0.8, 1.0, and 1.1 at [NaCl] = 1.0 M are
shown in Figure 9. Among these samples, B0 = 0.8 shows the
maximum viscosity (η0 = 1 Pa·s) whereas the viscosities at B0 =
1.0 and 1.1 are much lower (η0 < 0.01 Pa·s). Figure 9 shows
that the scattering intensity at low q is dramatically lower for
the B0 = 1.0 and 1.1 samples relative to the B0 = 0.8 sample.
The decrease in low-q scattering implies a shortening of the
micelles.53 The SANS data thus confirm that a transition from
long to short cylindrical micelles accounts for the viscosity
decrease beyond the peak in lecithin/bile salt mixtures. Figure
S4 shows the pair-distance distribution functions corresponding
to Figure 9 where the shortening in length of wormlike micelles
with increasing B0 can be clearly seen. Note that the intensity at
high q remains practically unchanged, indicating that the crosssectional size of the micelles is nearly the same for different B0.
3.3. Mechanism. In the preceding sections, we have shown
that with sufficient electrolyte concentration the addition of bile
salt can induce a growth of lecithin micelles in water. The
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electrolytes play a role in this regard. It has been shown that the
aggregation number of bile salt micelles in water increases with
increasing ionic strength due to the weakening of the
electrostatic repulsion between bile salts,56 which implies that
the curvature of the bile salt micelles is reduced at a high ionic
strength. Thus, with an increase in electrolyte concentration,
bile salt molecules no longer tend to favor the highly curved
end-caps of the cylindrical mixed micelles. This allows the
continuous growth of cylindrical micelles into chains that are
long enough to increase viscosity. As B0 exceeds B0max, the
excess bile salts may inevitably form stable end-caps that limit
the length of the micelles even at a high electrolyte
concentration. Also, more bile salts may further expand the
headgroup and reduce the effective packing parameter to a
value below 1/2 that favors more curved structures like
spherical micelles. This is why only in a narrow B0 range can we
tune the concentration of electrolytes to obtain highly viscous
or viscoelastic solutions of lecithin/bile salt mixed micelles.
Figure 10. Schematic of the self-assembled structures formed by
lecithin with and without bile salt in water. Lecithin is shown as a
molecule with a blue head and two red tails, while the bile salt is
represented as a facial surfactant. Lecithin alone in water has a critical
packing parameter ∼1 and tends to form bilayers. When bile salts are
incorporated, they bind to the lecithin headgroups in a back-to-back
form, thus expanding the head group area. This alters the net geometry
from a cylinder to a truncated cone and thereby induces the bilayers to
turn into cylinders. At a low ionic strength, the negatively charged
carboxylates or sulfonates of bile salts are highly repulsive, and thus
bile salts prefer to pack in the curved hemispherical end-caps of the
cylinders.
4. CONCLUSIONS
In this study, we have shown that mixtures of the physiological
lipids, lecithin and bile salt, can self-assemble into flexible
wormlike micelles in water. Micellar growth has been
demonstrated with four different bile salts. The wormlike
micelles are sufficiently long to entangle with one another and
form highly viscous and viscoelastic fluids. Such elongated
aggregates particularly occur under the following conditions:
(a) when the molar ratio of bile salt:lecithin is near equimolar
and (b) when a sufficient concentration of background
electrolyte is used. The transformation of the original lecithin
bilayers to cylindrical micelles upon the addition of bile salts is
attributed to a change in molecular geometry caused by the
binding of bile salt molecules to lecithin headgroups. The study
of these mixed micelles could provide insights into physiological processes involving lecithin, bile salt, and electrolytes. In
addition, these low-cost biocompatible viscoelastic fluids may
have potential for biomedical and cosmetic applications.
words, the end-caps of the cylindrical micelles are stabilized by
bile salts. When the end-caps are stable, the cylindrical micelles
lack a driving force for continued growth into long chains. With
more bile salts added into solutions, more end-caps are formed
and the cylinders are getting shorter. This explains why a high
B0 (beyond the peak in Figure 2) leads to the formation of
shorter cylindrical micelles, as confirmed by the SANS
experiments in Figure 9.
To obtain long wormlike micelles, B0 should be sufficiently
high to transform lecithin bilayers into cylindrical mixed
micelles (i.e., around 1), but it cannot be much higher because
it would lead to the formation of redundant end-caps. In the
present study, at B0max of each bile salt without the addition of
electrolytes, the amount of the bile salts is not enough to
transform the bilayers into micelles so that the samples are still
cloudy and of low viscosity, as shown in Figure 3 and Figure S3,
but this ratio is very close to the transition boundary. In a
micellar solution, the surfactants in micelles are in equilibrium
with the free surfactants in the bulk. It is known that the critical
micelle concentration (CMC) of ionic surfactants decreases
with increasing ionic strength because the repulsive forces
between surfactant headgroups are screened.44 The Hofmeister
salting-out effect of the added electrolytes also causes the CMC
of surfactants to decrease because the ions strengthen the
hydrophobic effect that drives the micellization in water.55 In
other words, when electrolyte concentration is increased, more
originally free surfactants self-organize into micelles.40,43
Following this scenario, the addition of electrolytes into
lecithin/bile salt solutions at B0max should force more originally
free bile salt molecules into micelles, which facilitates the
transformation of the bilayers into cylindrical micelles and turns
the solutions to be transparent.
The effect of electrolytes at B0max not only induces the
formation of cylindrical micelles from bilayers but also
promotes the growth of cylindrical micelles into long wormlike
micelles. To increase the length of the cylindrical micelles, the
end-caps must be destabilized. We suggest that the added
■
ASSOCIATED CONTENT
S Supporting Information
*
Zero-shear viscosities as a function of B0 for SDC, STC, and
STDC samples, dynamic rheology of SDC and STDC samples,
SAXS data and cryo-TEM image of STDC sample without the
addition of electrolytes, and pair-distance distribution function
corresponding to Figure 9. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected] (S.-H.T.).
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was financially supported by the grant of the
Ministry of Science and Technology, Taiwan (NSC 103-2923E-002-005-MY3). We acknowledge NSRRC, Taiwan, for
facilitating the SAXS experiments and NIST, USA, for SANS
experiments.
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Supporting Information for
Mixtures of Lecithin and Bile Salt Can Form Highly Viscous Wormlike
Micellar Solutions in Water
Chih-Yang Cheng†, Hyuntaek Oh‡, Ting-Yu Wang§, Srinivasa R. Raghavan‡, and ShihHuang Tung†,*
†
Institute of Polymer Science and Engineering, National Taiwan University, Taipei,
10617 Taiwan
‡
Department of Chemical and Biomolecular Engineering, University of Maryland,
College Park, MD 20742 USA
§
Instrumentation Center, National Taiwan University, Taipei, 10617 Taiwan
*Corresponding author. email: [email protected]
1. Zero-Shear Viscosity vs B0 for SDC, STC and STDC Samples
Figure S1. Zero-shear viscosity η0 as a function of the molar ratio of bile salt to
lecithin B0 for lecithin/bile salt mixtures in water. Lecithin concentration is fixed at
100 mM and NaCl concentrations of STC, STDC and SDC are 2, 0.75 and 1.5 M,
respectively.
2. Dynamic Rheology of SDC and STDC Samples
(b) STDC
(a) SDC
2
2
10
G',G'' (Pa)
G', G'' (Pa)
10
G"
1
10
0
10
G'
G"
1
10
G'
0
10
10
100
10
Frequency,  (rad/s)
100
Frequency,  (rad/s)
Figure S2. Dynamic rheology data of (a) lecithin/SDC mixture at B0 = 1.2 and
[NaCl] = 2.0 M, and (b) lecithin/STDC mixture at B0 = 1.0 and [NaCl] = 0.75 M in
water at 25°C. The lecithin concentrations of both samples are 100 mM. The plots
show the elastic modulus G′ and viscous modulus G″ as functions of frequency ω.
Both samples show liquid-like behaviors (G″ > G′) in the whole frequency range.
3. SAXS Data and Cryo-TEM Image of Lecithin/STDC Sample
(b)
(a)
0
-2
-1
Intensity, I (cm )
10
-1
10
-2
10
0.01
0.1
-1
Wave Vector, q(Å )
Figure S3. The self-assembled structure of lecithin/STDC sample at B0 = 1.0 without
the addition of electrolytes. The lecithin concentration is 20 mM. (a) The -2 slope in
SAXS data indicates bilayer structures. (b) Cryo-TEM image shows lamellae
stacked by the bilayers.
4.
Pair-Distance Distribution Function p(r) Corresponding to Figure 9
Figure S4. Pair-distance distribution functions p(r) obtained by IFT analysis of the
SANS data shown in Figure 9.